From yeast to humans, splicing is an essential step in the maturation of precursor messenger RNA (pre-mRNA). Anomalous pre-mRNA splicing can have lethal effects for the cell and has been linked to numerous human diseases such as cancer and neurodegenerative disorders. The spliceosome is a dynamic assembly of five small nucleolar RNAs (snRNA) and a large number of proteins that catalyzes splicing. Two snRNAs, U2 and U6, form the active site of the spliceosome. The structure of the U2/U6 complex has been the focus of much debate in recent years, because evidence has been presented for alternative conformations. We hypothesize that these conformations reflect different states of spliceosome activation, but specific structural dynamics information to support this hypothesis is still lacking. It is essential to study the structural dynamics of the U2/U6 complex to understand spliceosomal activation and catalysis, because this enzyme plays key roles in cell growth, differentiation and disease. We propose to use the powerful single molecule fluorescence technique to investigate these structural dynamics. We have previously demonstrated our approach to be particularly suited to elucidate the structural dynamics of RNA enzymes and reveal important information otherwise hidden in ensemble-averaged experiments. We aim at (1) revealing the U2/U6 conformational dynamics by single molecule fluorescence, (2) elucidating the role of Mg2+ ions in these dynamics, (3) linking these dynamics to spliceosomal activation in vivo, (4) comparing the structural dynamics of the U2/U6 complex from humans and yeast and (5) elucidating the role of spliceosomal protein Prp24 in spliceosomal activation. PUBLIC HEALTH RELEVANCE: From yeast to humans, splicing is an essential step in the maturation of messenger RNAs that are used to synthesize functional proteins. Anomalous splicing can have lethal effects for the cell and has been linked to numerous human diseases such as cancer and neurodegenerative disorders. The spliceosome is a large RNA-protein complex that catalyzes splicing. The structure of the catalytic center of the spliceosome has been a matter of debate in recent years because of its dynamic nature. It is essential to investigate the structural dynamics of the catalytic core of the spliceosome to understand its biological function, because this enzyme plays key roles in cell growth, differentiation and disease. We propose to use the powerful single molecule fluorescence technique to resolve these structural dynamics and characterize their mechanism in unprecedented detail.